Ondertepoort Journal of Veterinary Reearch Volume 9, Number, December, 9 The Government Printer, Pretoria URESE TEST FOR CHRCTERIZING BRUCELL STRINS G. C. VN DRIELEN, Veterinary Reearch Intitute, Ondertepoort any train of Brucella, notably the merican Br. ui type are capable of hydrolyzing urea to form C0 and NH The reaction can proceed to the formation of ammonium carbonate. The method at preent in ue for meauring ureae activity to Brucella train are either rather inaccurate or require pecialized equipment (Wohlfeil & Weiland, 97; Piihel, 9; Ferguon & Hook, 9; Schneider & Gunderon, 9; Chritenen, 9; Bauer, 99; Hoyer, 950 ; Pacheco & Thiago de ello, 950; Sander & Warner, 95; Renoux & Quatrefage, 95; Huddleon, 95; Pickett, Nelon & Liberman, 95; Niznanky & Kremery, 95; Godgluck & arggraff, 955). In addition they expoe worker to infection with virulent material, e.g. the ue of the Warburg apparatu, or Van Slyke & rchibald' (9) titrimetric method or Conway' (99) microdiffuion method. For thee reaon the Joint FO/ WHO Expert Committee on Brucelloi feel that if the ureae tet i to be of real value, it hould be improved (95). In thi report the work carried out at the Ondertepoort centre leading to the intitution of a routine ph threhold ureae tet, will be recorded. Thi tet i reliable and the reult can be preented numerically. Strain TERILS ND ETHODS (a) Reference train : Br. me/iteni (Beltville) 5 Br. abortu (Weybridge) 0 Br. ui (merican) (inneota) (b) Laboratory train: S. 9 Br. abortu (Beltville) Rev.. Br. meliteni (Elberg, Berkeley) ----- - - ----------------------- -- Received for publication on November, 9.- Editor 5
URESE TEST FOR CHRCTERLZ LNG BRUCELL STRLNS Typing edia (c) Field train: recent iolate from cae including: man............................ Br. me!iteni Br. abortu cattle........................... Br. abortu goat........................ Br. abortu heep (ram)............. Br. ovigenitalium heep (ewe).............. Br. abortu ovi The train were typed on the following characteritic: () Hot an, ox, heep, goat () Staining reaction Hanen' (Hanen et al., 99) Gram' (Van Drimmelen, 90) Stamp-cEwen' (Stamp et al., 950) () ntigenic tructure onopecific agglutination: meliteni, abortu (Jone, 958) () Bacteriophage en itivity Lyi by ahortu phage, ui phage (Parna et al., 958 ; Van Drimmelen, 90 : Jone, 90) (5) Biochemical propertie Dye enitivity (Pickett et al. 95) H S production (Huddleon,97) Di-ethyl-di-thio-carba mate reitance (Renoux, 95) () Cultural character C0 dependence (Huddleon, 9 l) S- R variation: acriflavine tet (Braun et al., 97) ; heat agglutination (7) Biological propertie Pathogenicity for guinea pig The urea medium of Rutigian & Stuart (9) wa prepared a nd ued a recommended by Pickett, Nelon & Li berm an ( 95) at ph. The modification introduced for the preent work wa adjutment to ix different ph value : - 0 (with a difference of one unit from one tube to the next). Teting Ureae activity wa teted by adding 0 5 ml of a dene upenion (about 0 9 organim from a 8 hour urface culture on albimi agar) in aline to a erie of tube each containing 0 ml of medium at the variou ph value. fter being agitated the tube were incubated at 7 C. The reult were read after four hour and confirmed after 8 hour. Reult were recorded a the "ph threhold value" i.e. the lowet ph at which ureae activity wa indicated by indicator colour change. 5
G. C. VN DRIELEN Control tet Tet by the method of Schneider & Gunderon (urea olution) (9), Chritenen (urea agar) (9), Pacheco & Thiago de ello (paper trip) (950), and titrimetric H S0 tet (Ondertepoort) and alo titration of oybean ureae were carried out for comparative purpoe. RESULTS On the bai of ph difference in media a recommended by earlier worker, the enitivity of ureae wa invetigated by firt determining the optimum ph for maximum activity of a known amount of ureae. The :finding are hown in Table. T BLE.- Titrimetric determination of the activity of oybean ureae at different ph level : The percentage of urea converted by a contant amount of ureae during three hour at room temperature N Urea Urea ued Gm ph titrated calculated Gm Gm --- - - - Percentage recovery 0............. 8 7 8 89 0................. 9 9 8 99 0........ 7 9 7 0 85 0.............. 7 77 8 0............. 0 7 8 5 78 Different dilution of oybean ureae were then teted in et of tube at ix different ph level containing urea broth with indicator. The reult after four hour and 8 hour were identical and are hown in Table. TBLE.-ctivity of oybean ureae contained in one tablet and ufficient to hydrolize 80 mgm urea, diluted erially and then teted at ix ph level in the medium ued for the Bruce/la ureae tet ph................................................. 0. """......... 0....... Undi- I luted l : I : Ureae dilution :8 l : l : I l : l : 8 5
URESE TEST FOR CHRCTERIZING BRUCELL STRINS The higher dilution were found to have a limited range of activity. The threhold value for undiluted ureae wa ph 0 wherea for ureae diluted in it wa ph. The ureae concentration could therefore be meaured by the lowet ph at which urea wa broken down i.e. the " ph threhold value ". The ureae activity of Brucella tock train wa teted at weekly interval by adding uniform upenion of ucceive culture to et of urea tet media a decribed. The reult are ummaried in Table. T BLE.- Ureae activity of approximately 0 9 organim of different tock train of Bruce/la a hown by the ph threhold tet Strain Date 5 0 Sl9 0 09 ph threhold.ay.0...........,,.0............ 8.,,. 0..................June.0..............,,.0........... 0.,,.0................ 9.,,. 0.........July. 0.............,,.0..................,,. 0................. 0.ug.. 0............. 7.,,.0...............,,.0..............,,.0..................Sept.. 0............. 0 0 0 0 0 No activity Not teted The ph threhold value for each train wa found to be reaonably contant and the reult were reproducible. The technique ha accordingly been applied to routine teting of South frican field train at Ondertepoort. Characteritic of FO/ WHO reference train and of new iolate from Southern frica are compared with each other when arranged in order on the cale of their ureae tet reult a hown in Table. further record of reult obtained by thi tet, i illutrated in a lit of ome train examined. See Table 5. 5
TBLE.- Type of Brucella organim f ound in South frica a grouped bv the ureae tet compared with Brucel!a reference train Legend : = abortu; S = ui; = meliteni; T = thionin ; BF = baic fuchin ; - = reaction negat ive ; to = reaction poiti ve. 0 0 < 0 c: tt'i I Br I I S.. S.. Bruce/la S.. Bruce/la WHO S.. Br. ovigenitalium WHO. u i Brucella Brucel!a abortu meliteni Bruce/la Bruce/la Stram I WHO abortu abortu - - melt!en- abortu 0 Sl9 OVIS I I SIS I I' 5 J 09 0 5 0 0 0 05 0 00 0 I --- - - -----,- i I I. Hot Pig Cattle Cattle I Cattle I Human Human I Goat ram ram r r:._m Cattle Virulence......... I I Serology.......... Phage.......... S - - I - - - co,......... - - ' Dye enitivity- T........... - I I - RF......... - - - - - H S..............
Di-ethyl- Ureae Virudi-t hio activity lence for carba- by ph guiflea mate threhold pig reaction method mm Legend : S = mooth ; l = intermediate ; R = rough ; = mucoid ; = " abort u "; S = " ui " ; = " meliteni "; - = reaction negative;. = tet not completed. Numeral indicate the ize (radiu in mm) of the inhibited zone round dye ta blet and D.E.D.T.C. tablet; the ph threhold in the cae of ureae ; day in the cae of H S ; a nd the degree in guinea pig leion. (8) 7 9 0 8 J 7 JO 9 9 0 0 c::: m en > tn ;j >r 0 () ;:c; >- N z Cl t:d c:: () tn r >- en...; >- z en TBLE 5.- Characteritic of ome South frican train of Brucella organim = ====-- --- - Strain reference number Refer- ' Type or ence Colonie CO, hot number R depenpecie or r ' dence pecimen I or -. I ono- Dye inhibition Bacteno- pecific phae aggluti-.. I Baic ly I nation Thonme Fuchm or S or mm mm H,S production l,, or day V\ 0\ Control- 0............. 0................ 58................ 0 8................. 0 7.......... 58 0............ South frican train- H. 0............ 0. 0.............. 0. 0............... 0. 0 5.............. 0. 0.............. 0. 0 9......... 0. 0.IO......... B. 0.............. B. 0 5........ 0. 0!......... H. 0............. -f. 0............ 0. 0............ Hu:an I Cattle Cattle I Human Human 5 0 9 Parna Rev. I blood emen emen emen emen mi lk emen foetu foetu foetu blood blood emen l [ l I I l 5 l 8 5 5 8
G. C. VN DRIELEN DISCUSSION Ureae tet previouly decribed depend on the rate of change of ph. Thi depend not only on the concentration of ureae but alo on the compoition of the medium, temperature and agitation. The rate of ph change i therefore not a reliable meaurement of ureae activity. The ph threhold method give more reproducible reult. ha been hown, the lowet ph at which ureae activity can be initiated i directly correlated to the concentration of ureae preent. Once the reaction ha tarted, the ph rie and condition become progreively more favourable thu enuring optimum activity. The ph threhold method therefore give an " all or none " repone which i more or le independent of time and which can be recorded numerically. ll ureae are uually conidered to be identical. Should ureae from different ource prove to differ in regard to optimum ph value, the ph threhold tet could be ued to characterie ureae. In thi cae it might be found that the ureae of Br. ui i particularly reitant to inhibition by low ph. The preent work how that ureae activity i an independent character of train of Bruce/la organim, not correlated with other characteritic. It would appear that South frican train of Br. abortu diplay a fairly high, though variable, ureae activity, comparable to that of Br. meliteni in other part. CKNOWLEDGEENTS Permiion from the Chief, Veterinary Reearch Intitute, to publih thi report and technical aitance from r. F. D. Horwell, r. P. V. ulder and i J.. Dreyer are incerely appreciated. REFERENCES BUER, H. (99). tudy of Brucella and Proteu ureae. PhD-Thei Univerity of inneota. BRUN, W. & BONESTELL,. E. (97). Independent variation of characteritic in Brucella abortu variant and their detection. m. J. Vet. Reearch, Vol. 8, pp. 8-90. CHRISTENSEN, W. B. (9). Urea decompoition a a mean of differentiating Proteu and Paracolon culture from each other and from Salmonella and Shigella type. J. Bact., Vol. 5, pp. -. CONWY, E. H. (99). icrodiffuion analyi and volumetric error. Edit. London : Croby Lockwood, and Son Ltd. FERGUSON, W.W. & HOOK,. F. (9). Ureae activity of Proteu and Salmonella organim. J. Lab. Clin. ed., Vol. 8, pp. 75-70. GODGLUCK, G. & RGGRFF, I. (955). Die Ureae-ktivitat der Brucella-Bakterien. Zbl. Vet.-ed., Vol., pp. 5-5. HNSEN, K. & KOSTER, H. (99). Nachwei von Brucella abortu (Bang) auf Grund der alkalifetigkeit. D.T. W., Vol., pp. 79-70. HOYER, B. H. (950). Some apect of the phyiology of Brucella organim. Brucelloi, m.. dv. Sci. Wahington D.C. 9-5. HUDDLESON, I. F. (9). Studie in infectiou abortion. J. m. Vet. ed. oc., Vol. 0, pp. 5-5. HUDDLESON, I. F. (97). biochemical method of differentiating Brucella abortu from Brucella meliteni-parameliteni. J. Bact., Vol., p.. HUDDLESON, I. F. (95). Brucelloi in man and animal. The Commonwealth Fund, New York rd ed. 57
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